Team:Sumbawagen/Notebook/protocol1

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iGEM Sumbawagen 2014 · Econey

Notebook – Protocol – 1. Primer design

1.DNA sequence of E.coli adenylate cyclase (cyaA; accession code NC_000913 REGION: 3991153..3993699; 2547 bp) and E. coli IIA(Glc) (crr; accession code J02796; 510 bp) were obtained from Genebank http://ncbi.nlm.nih.gov/

2.Both DNA sequences were checked for the “in availability” of restriction sites of EcoRI (G^AATT_C), XbaI (T^CTAG_A), SpeI (A^CTAG_T), and PstI (C_TGCA^G) by Bioedit program using menu: Sequence/Nucleic Acid/Restriction Map/Generate Map.

3. Forward primers were designed to contain (from 5’ direction) :

  • additional sequence to accommodate restriction enzyme recognition; TTA
  • sequence from backbone pSB1C3 which has both EcoRI and XbaI sites; GAATTCGCGGCCGCTTCTAGA
  • seven codons of the initial part of the genes which include start codon

4. Reverse primers were designed to contain (from 5’ direction) :

  • additional sequence to accommodate restriction enzyme recognition; TTA
  • sequence from backbone pSB1C3 which has SpeI site; ACTAGTA
  • double stop codon (in reverse complement sequence); TTATTA
  • six codons of the last part of the genes (in reverse complement sequence)
  • For cyaA, besides the last part of the genes which codes for full length sequence, other reverse primer was also designed to contain the last part of N-terminal catalytic domain (full length sequence, 2547 bp; N-terminal catalytic domain, 1605 bp)

Resulted primers sequences were as follow

  • cyaA full length forward primer (primer name “ACEF”)
  • 5’-TTA GAA TTC GCG GCC GCT TCT AGA TGT ACC TCT ATA TTG AGA CTC TG-3’
  • cyaA catalytic domain reverse primer (primer name “ACER1”)
  • 5’-TTA ACT AGT ATT ATT ATT CCG AGA GAT CGG GTG AAA T-3’
  • cyaA full length reverse primer (primer name “ACER2”)
  • 5’-TTA ACT AGT ATT ATT ACG AAA AAT ATT GCT GTA ATA G-3’
  • crr forward primer (primer name “AEF”)
  • 5’-TTA GAA TTC GCG GCC GCT TCT AGA TGG GTT TGT TCG ATA AAC TG-3’
  • crr reverse primer (primer name “AER”)
  • 5’-TTA ACT AGT ATT ATT ACT TCT TGA TGC GGA TAA C-3’